Generation of marmoset primordial germ cell–like cells under chemically defined conditions

Marmoset primordial germ cells (PGCs) express SOX17, AP2Ɣ, and BLIMP1 in vivo, and under specific feeder-free conditions, marmoset iPSCs can be induced into PGC-like cells in vitro.

We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): In this manuscript, Kurlovich et al refine current methodologies to generate marmoset PGC-like cells.They first generate new feeder-free marmoset iPSC lines, which then they use as a starting point to improve conditions for PGC-like cell specification.The method, tools, and improvements described in the manuscript will be useful for the community.The manuscript is well written, the results are clear, and the conclusions are supported by the data.I support the publication of this manuscript once the following comments have been addressed: 1.The authors find out that removing Activin-A from the pre-induction step is favourable.However, this was done only using the expanded potential conditions.Is it possible to obtain PGC-like cells from the conventional primed cultures if Activin-A is removed during pre-mesoderm differentiation?Does Activin-A bias primed cells towards mesoderm?2. The identity of the cells cultured under expanded potential conditions is not clear.The authors claim they are naïve-like, but Figure 4A shows the expression of endoderm markers.Could the authors compare the transcriptome of their cells to available datasets of human PSCs cultured under different regimes (i.e., naïve, formative, primed, expanded potential)?
Minor comments: -Please indicate conditions of imaging (e.g., microscope and objectives used) -Figures 4B and 4C should be quantified.
Reviewer #2 (Comments to the Authors (Required)): Julia and her colleagues investigated the induction of primordial germ cell-like cells (PGCLCs) in marmosets.They observed that culturing cjiPSCs in cjPSCM, as opposed to other mediums, maintained a more naïve pluripotent stem cell state and ensured PGC competence for subsequent differentiation.Additionally, the removal of ActA from the preME differentiation medium appeared to increase the number of cjPGCLCs. Comments: (1) The paper conducted RNA-Seq of cjiPSCs, pre-ME, and FACS-sorted cells, examining marker genes to illustrate cell identity.However, this doesn't fully support the conclusion of a "transcriptome similar to human PGCs/PGCLCs" as stated in the abstract.The authors should leverage the RNA-Seq data for a comprehensive transcriptomic comparison between marmoset, mouse, and human, given the availability of public data for the latter two.
(2) For the cjPSCM, the authors proved that cjiPSCs in cjPSCM is more naïve compared to other medium.This is different from human since human starts in a primed state and it is the intermediate state that shows the dual property of naïve and primed properties (PMID: 31875561; PMID: 36640324).I suggested comparing the naïve gene expression levels in marmosets and humans in their differentiation processes including iPSC and PreME stages.
(3) I strongly recommend the authors include a schematic differentiation figure that outlines medium selection, differentiation steps, and outputs, providing a clearer visual representation of the process.
(4) Consider changing the word "upregulate" to "express" in the subtitle "cjPGCLCs upregulate the germ cell-specific transcriptional program."As the title suggests a program, should be defined as a gene list with a specific number of genes.The authors could define this gene list and use a gene enrichment score to demonstrate it effectively.
(5)From page 6 please state clearly which one cjiPSCs (DPZ_cjiPSC#1-6) was used, and if there is any difference of cell lines in PGCLC differentiation efficiency?Similarly, for the test of the medium, did the medium have the same effect on all lines, or did the author test it only in one cell line?We would like to sincerely thank the reviewers for their constructive comments, which helped to significantly improve our paper.Please find our point-by-point response (in blue) below.
Changes in the text of the paper are highlighted in green.

Reviewer #1:
In this manuscript, Kurlovich et al refine current methodologies to generate marmoset PGClike cells.They first generate new feeder-free marmoset iPSC lines, which then they use as a starting point to improve conditions for PGC-like cell specification.The method, tools, and improvements described in the manuscript will be useful for the community.The manuscript is well written, the results are clear, and the conclusions are supported by the data.I support the publication of this manuscript once the following comments have been addressed: 1.The authors find out that removing Activin-A from the pre-induction step is favourable.However, this was done only using the expanded potential conditions.Is it possible to obtain PGC-like cells from the conventional primed cultures if Activin-A is removed during premesoderm differentiation?
To address this question, we have differentiated cjiPSCs cultured in conventional primed conditions (TESR) into precursors of mesendoderm (pre-ME) without Activin A (ActA) and subsequently into cjPGCLCs.Immunofluorescence analysis of embryoid bodies (EBs) shows that the PGC markers SOX17, AP2Ɣ and BLIMP1 are expressed in a low number of cells (Revision Fig. 1).However, their expression does not appear to colocalise, which is similar to the results obtained with the addition of ActA (Fig. S2C).These results suggest that cjiPSCs cultured in primed conditions do not efficiently differentiate into PGCLC-competent pre-ME, regardless of the presence/absence of ActA.We have included these data in the manuscript (Fig. S5A).
Revision Fig.

Does Activin-A bias primed cells towards mesoderm?
We have now performed RNA-seq with pre-ME differentiated with (pre-ME+ActA) or without ActA (pre-ME-ActA) from cjiPSCs in primed conditions (TESR).Differential gene expression analysis (DESeq2; padj < 0.05, log2foldchange >2) shows significant upregulation of 176 genes in pre-ME+ActA compared to pre-ME-ActA (Revision Fig. 2A).As predicted by the reviewer, many of these genes are associated with mesoderm development, including EOMES, SNAI1, GSC, HAND3, WNT3 (Revision Fig. 2B).Consistently, gene ontology (GO) classification with upregulated genes shows enrichment for terms such as muscle tissue development, pattern specification process, gastrulation, and mesoderm development (Revision Fig. 2C).Taken together, these results indicate that ActA primes pre-ME for mesoderm fate during differentiation from cjiPSCs cultured in conventional primed conditions.We have included this data in (Fig. S5B-C).2. The identity of the cells cultured under expanded potential conditions is not clear.The authors claim they are naïve-like, but Figure 4A shows the expression of endoderm markers.
Could the authors compare the transcriptome of their cells to available datasets of human PSCs cultured under different regimes (i.e., naïve, formative, primed, expanded potential)?
We agree with the reviewer that this conclusion needs to be revised.Indeed, we observe both the upregulation of some markers of naïve pluripotency and somatic differentiation in cjiPSCs cultured in cjPSCM versus TESR.As suggested by the reviewer, we compared the transcriptomes of our condition to available published datasets of human pluripotent stem cells (hPSCs) in naïve (E-MTAB-5114), formative (GSE131556), expanded potential (E-MTAB-7253) and primed conditions (E-MTAB-7253).First, we performed global transcriptome comparisons using principal component analysis (PCA; Revision Fig. 3A).hPSC samples are separated across the pluripotency spectrum.In particular, naïve hPSCs are distant from all other samples, whereas primed hPSCs are separated from formative and expanded pluripotent hPSCs along PC2.Interestingly, we observed a comparable shift of cjiPSCs cultured in cjPSCM from cjiPSCs in TESR.However, it should be noted that cjiPSCs are generally quite distant from hPSC samples, which is likely due to technical differences between RNA-seq datasets from different studies, but also species-specific differences.To analyse this further, we compared the gene expression profile of markers of pluripotency and differentiation between marmoset and hPSC samples.Interestingly, similar to formative and expanded pluripotent hPSCs, cjPSCs in cjPSCM upregulate a subset of genes associated with naïve and formative pluripotency including ANPEP, PECAM1, POU5F1, NANOG, KLF5, DPPA3, NLRP7, DPPA3, KLF5, OTX2 and TFAP2C as well as primed pluripotency including FAT3, THY1, and SOX11 (Revision Fig. 3B).However, there are also notable differences including the upregulation of SOX17 and FOXC1 in cjiPSCs.Taken together, these results indicate that cjPSCM induces gene expression changes in cjiPSCs that may lead to a partial reversion from primed to a formative-like pluripotent state.We have included this analysis in the manuscript (Fig. 4B and Fig. S3C).

Minor comments:
-Please indicate conditions of imaging (e.g., microscope and objectives used) We have included information on imaging in the Methods section.
-Figures 4B and 4C should be quantified.
We have quantified the immunofluorescence images shown in Fig. 4B/C, which shows 6.9% (+/-1.51%)cjPGCLCs induced from pre-ME+ActA as compared to 9.9 (+/-2.65%)cjPGCLCs induced from pre-ME-ActA.We have included this information in the manuscript.
Reviewer #2 (Comments to the Authors (Required)): Julia and her colleagues investigated the induction of primordial germ cell-like cells (PGCLCs) in marmosets.They observed that culturing cjiPSCs in cjPSCM, as opposed to other mediums, maintained a more naïve pluripotent stem cell state and ensured PGC competence for subsequent differentiation.Additionally, the removal of ActA from the preME differentiation medium appeared to increase the number of cjPGCLCs. Comments: (1) The paper conducted RNA-Seq of cjiPSCs, pre-ME, and FACS-sorted cells, examining marker genes to illustrate cell identity.However, this doesn't fully support the conclusion of a "transcriptome similar to human PGCs/PGCLCs" as stated in the abstract.The authors should leverage the RNA-Seq data for a comprehensive transcriptomic comparison between marmoset, mouse, and human, given the availability of public data for the latter two.
Next, we compared the gene expression profile of a comprehensive list of PGC markers between PGCLC samples, including embryonic human PGCs (hPGCs, week7, GSE159654) (Revision Fig. 4B).A large number of genes show a similar expression profile in all three species including the expression of POU5f1 (OCT4), NANOG, PRDM1, TFAP2C, and KIT.Notably, PRDM14 could be only detected in hPGCLCs and mPGCLCs, indicating speciesspecific differences.Importantly, we also observed primate-specific upregulation of PGCassociated genes including SOX17, KLF4, TFAP2A, GATA3, and TBX3 in hPGCLCs and page of 9 5 cjPGCLCs as opposed to mPGCLCs.Taken together, these data suggest that the transcriptional profile of cjPGCLCs is reminiscent of that of hPGCLCs.We have included this analysis in the manuscript (Fig. 7).

Revision Fig. 4: The transcriptional programme of mPGCLCs, cjPGCLCs and hPGCLCs. (A) Pearson correlation of RNA-seq datasets for d4 cjiPGCLCs induced from pre-ME (+/-ActA) and published data for d4/d6 mPGCLCs and d4 hPGCLCs. (B) Heatmap showing expression levels of genes associated with PGC development. Scale: Log2(normalised counts+1).
(2) For the cjPSCM, the authors proved that cjiPSCs in cjPSCM is more naïve compared to other medium.This is different from human since human starts in a primed state and it is the intermediate state that shows the dual property of naïve and primed properties (PMID: 31875561; PMID: 36640324).I suggested comparing the naïve gene expression levels in marmosets and humans in their differentiation processes including iPSC and PreME stages.
Please note that we have revised our conclusion regarding the pluripotent state of cjiPSCs cultured in cjPSCM (please see Reviewer#1 point2 above).
Following the reviewer's suggestion, we analysed the expression levels of genes associated with naïve and primed pluripotency using published RNA-seq datasets for primed hESCs (GSE159654) and human pre-ME (GSE159654) and our datasets for cjiPSCs and marmoset pre-ME.Indeed, human and marmoset pre-ME share the expression of subsets of genes associated with naïve and primed pluripotency.This is consistent with the aforementioned previous study (PMID: 36640324 1 ), which shows by PCA that the global transcriptome of human pre-ME is close to that of primed hESCs (Fig. 1H   expression features of naïve and primed pluripotency.We have included this analysis in the manuscript (Fig. S7).

Revision Fig. 5: Expression profile of genes associated with pluripotency in pre-ME. Heatmap showing expression levels of genes associated with naïve and primed pluripotency. RNA-seq datasets are from primed hESCs (GSE159654), human pre-ME ((h)pre-ME, GSE159654), cjiPSCs (this study) and marmoset pre-ME ((cj)pre-ME, this study). Scale: Log2(normalised counts+1).
(3) I strongly recommend the authors include a schematic differentiation figure that outlines medium selection, differentiation steps, and outputs, providing a clearer visual representation of the process.
We have included schematics of the differentiation approaches in the relevant figures and summarised this in Fig. S8.
(4) Consider changing the word "upregulate" to "express" in the subtitle "cjPGCLCs upregulate the germ cell-specific transcriptional program."As the title suggests a program, should be defined as a gene list with a specific number of genes.The authors could define this gene list and use a gene enrichment score to demonstrate it effectively.
Following the reviewer's recommendation, we changed the subtitle to 'cjPGCLCs express genes associated with PGC fate'.We also performed gene set enrichment analysis (GSEA) with a defined set of PGC genes between cjPGCLCs and somatic cells, which confirmed a significant enrichment of PGC genes in cjPGCLCs (Revision Fig. 6).However, we have decided not to include this analysis in the manuscript, since we think that the newly added analysis (Revision Fig. 4) already shows the expression levels of individual PGC genes in cjPGCLCs compared to human and mouse PGCLCs.(5) From page 6 please state clearly which one cjiPSCs (DPZ_cjiPSC#1-6) was used, and if there is any difference of cell lines in PGCLC differentiation efficiency?Similarly, for the test of the medium, did the medium have the same effect on all lines, or did the author test it only in one cell line?
We observed the induction of AP2Ɣ-BLIMP1 clusters at d2 of differentiation.However, at d6 of differentiation, only a few BLIMP1-positive cells remained, which appeared to be mutually exclusive with SOX17-expressing cells.This suggests that the cells adopted different cell fates during the time course of differentiation, which is consistent with the results obtained for DPZ_cjiPSC#2.
We also adapted the culture of DPZ_cjiPSC#3 and DPZ_cjiPSC#5 to cjPSCM.We then differentiated cjiPSC lines into pre-ME with or without Activin A and induced cjPGCLCs, resulting in clusters of SOX17-expressing cells (Revision Fig. 7B).In addition, a subset of these cells was co-expressing BLIMP1, which appeared to be more efficient in conditions where pre-ME was differentiated without Activin A. Thus, these results suggest that cjPSCM can be used to culture at least three independent cjiPSC lines for subsequent cjPGCLC differentiation.We have added this data (Fig. S1 and S4) and clarified the information about the cell lines in the manuscript.Thank you for submitting your revised manuscript entitled "Generation of marmoset primordial germ cell-like cells under chemically defined conditions".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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RevisionFig. 6 :
Gene set enrichment for genes associated with PGCs.Gene set enrichment analysis (GSEA) shows significant enrichment of a defined set of PGC genes in d4 cjPGCLCs induced from pre-ME (+/-ActA) compared to somatic cells.
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Revision Fig. 2: Activin A primes pre-ME differentiated from primed cjiPSCs for mesoderm fate. (A)
Differential gene expression analysis (DESeq2) of RNA-seq datasets of pre-ME differentiated with or without ActA (+/-ActA) from cjiPSCs cultured in TESR.(B) Heatmap showing expression levels of genes associated with mesoderm development in pre-ME.r: replicate.(C) GO term enrichment for genes significantly upregulated in pre-ME -ActA compared to pre-ME +ActA.
Chen et al. 2019t al., 2023 1 ).Furthermore, we observe an upregulation of TFAP2A in marmoset pre-ME -ActA, which is associated with the developmental trajectory of hPGCLC fate, as shown byChen et al. 2019 (PMID: 31875561 2 ).Taken together, our data suggest that marmoset pre-ME harbour gene